ABSTRACT Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), an obligate human pathogen whose persistence within the human population depends on the ability to drive successive cycles of transmission, infection, and disease. Consistent with its importance in maintaining elevated TB prevalence rates, there is renewed interest in the potential for novel interventions to interrupt Mtb transmission in endemic regions. While attractive, this approach requires rapid, reliable, and scalable technologies to detect live bacilli released by Mtb- infected individuals independent of TB symptoms. This is a challenging undertaking given the practical and biosafety difficulties posed by working with often paucibacillary samples of an infectious pathogen increasingly associated with drug resistance. For this reason, most studies to date have focused on smear-positive TB disease, thereby ignoring the potential contribution of sub-clinically infected individuals (i.e., those who are minimally symptomatic or even asymptomatic) to Mtb transmission. In attempting to address this limitation, we recently described the development and preliminary validation of the Respiratory Aerosol Sampling Chamber (RASC), a personal clean-room equipped with advanced, high-efficiency filtration, sampling, and particulate detection technologies that allows biosafe, non-invasive capture and isolation of live Mtb bacilli from confirmed TB patients during normal respiratory activity; that is, without the requirement for induced cough. Building on the postulate that the number of viable Mtb bacilli produced per unit of exhaled bioaerosol provides a reliable measure of TB transmission potential, we propose here to optimize the RASC platform for rapid, semi- quantitative detection of viable Mtb bioaerosols in large numbers of individuals. Existing, well-defined cohorts of subjects will be screened to identify the proportion and infectiousness of Mtb transmitters and, importantly, will be extended beyond smear-positive TB cases ? a first for direct studies of Mtb transmission. Results will be stratified by HIV status and used to model the contribution of sub-clinical Mtb transmission to TB prevalence rates, in turn informing the design of implementable interventions to reduce the TB burden in endemic TB/HIV settings. Finally, this approach also suggests the potential to use the RASC to understand the impact of TB chemotherapy on Mtb bioaerosol release.